It is known that a greater sensitivity can be achieved in a fluoroimmunoassay, if the competitive reactant is heavily labelled. However, as the number of fluorophores bound to such reactant is increased, the spacing between adjacent fluorophores decreases until a critical distance of 60 .ANG. to 100 .ANG. is reached. At this critical distance, or less, the fluorophores are self-quenching, i.e., the fluorescent level becomes less than an equivalent number of fluorophore molecules distanced greater than 100 .ANG.. Quenching also occurs in certain assays, e.g., for thyroid hormones, because of the iodine present in such materials. In the past, fluorometric assays for these reactants have been extremely difficult.
The invention heavily labels a competing ractant with fluorophores, which may be subjected to self-quenching, and then the fluorophores are separated from the reagent after competitive binding has occurred. Following separation, the fluorophores are spaced beyond the critical distance, whereby self-quenching ceases and the available fluorescent level is restored. Accordingly, a more sensitive fluoroimmunoassay can achieved.